QTY FC Fusion Receptor Proteins

ABSTRACT

The present invention is directed to QTY CCR9 and CXCR2 variant Fc receptor fusion proteins, methods for the preparation thereof and methods of use thereof.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/002,666 filed Mar. 31, 2020 and U.S. Provisional Application No.63/048,730 filed Jul. 7, 2020. The entire teachings of theabove-referenced applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Chimeric antigen receptor (CAR) T-cell therapy is a cellularimmunotherapy in which patient's T cells are engineered in vitro totarget and eliminate cancer cells in vivo. In CAR-T treatment, the Tcells from a patient's blood are extracted by apheresis. The gene for aspecific receptor (CAR) which binds to a certain tumor target isdelivered to the T cells by viral vector or non-viral transposon methods(Ittershagen et al., 2019; Jain & Davila, 2018; Srivastava & Riddell,2015). At present, two anti-CD19 CAR-T products have been approved bythe US FDA for the treatment of B-cell acute lymphoblastic leukemia andnon-Hodgkin lymphoma; CAR-T therapy for other cancer types undergoingvigorous clinical studies. CAR-T therapy holds great promise fortreating hematologic malignancies, and recent clinical evidence hasindicated that similar approaches can also be used to treat solid tumors(Baybutt et al., 2019).

However, there are several side effects during CAR-T treatment that ispotentially fatal to the patients, including: cytokine release syndrome(CRS), neurologic events, neutropenia and anemia (Xu & Tang, 2014).Among all the side effects, CRS is considered as a significant one thatcan be life-threatening. Cytokines are immune mediators essential inmany bodily actions in human. Yet, a large and rapid release ofcytokines into the blood from immune cells can induce “cytokine storm”(“CRS”). Most patients with CRS develop a mild flu-like reaction such asfever, fatigue, headache and rash. However, the reaction may progress toan uncontrolled, systemic inflammatory response with extreme pyrexia andbecome life-threatening (Shimabukuro-Vornhagen et al., 2018).

Manifestation of CRS can also be triggered by bacterial and viralinfections such as influenza and hepatitis virus (de Jong et al., 2006;Savarin & Bergmann, 2018; Tisoncik et al., 2012). The current COVID-19(Coronavirus Disease 2019) global pandemic involves CRS in many stagesof its pathological course that causes lung fibrosis, acute respiratorydistress syndrome, and eventually leads to multiple organ failure (Huanget al., 2020; Xu et al., 2020). Other conditions, includinggraft-versus-host disease, sepsis, Ebola, avian influenza, smallpox, andsystemic inflammatory response syndrome, also involve extensive releaseof undesired cytokines (Drazen, 2000).

Fc fusion proteins, an immunoglobulin Fc region directly linked to, forexample, an extracellular domain of a receptor, are therapeutic agentsthat can bind and eliminate ligands. An example of a fusion protein isetanercept, an anti-TNF drug currently marketed as a treatment for avariety of inflammatory diseases. Although the therapeutic protein hasbeen found to be safe and effective, the utilization of the molecule islimited by an unexpected short serum half-life (80-120 hours about 4days) when administered by subcutaneous injection. Other attempts todevelop Fc fusion proteins to clear aberrant protein expression has beenlimited. However, improved strategies for Fc fusion protein design isneeded. Early treatment of sepsis, for example, includes the use oftimely, appropriate antibiotics, intravenous fluids, oxygen therapy aswell as vasopressor and inotropic support where needed. Other additionaltreatments including extracorporeal or so-called blood purificationtechniques (BPT) have been tried (Zhou et al., Blood Purification andMortality in Sepsis: a meta-analysis of randomized trials. Crit. CareMed. 2013; 11, 507-13. These techniques include (among others):hemofiltration, hemoperfusion, intermittent or continuous high-volumehemofiltration, plasmapheresis or adsorption. The rationale behind suchan approach is to achieve “immune homeostasis” which theoreticallyreduces the potential danger caused by dysregulation of the hostresponse to infection. This may be heralded by a profound rise ininflammatory mediators, including cytokines which contribute to thedramatic systemic effects of sepsis, mainly in septic shock.

SUMMARY OF THE INVENTION

The application provides novel Fc fusion water soluble GPCR proteinswith QTY membrane regions.

The applicants have previously devised a novel tool called “QTY code”which regulates the water solubility of redesigned membrane proteinsthrough pairwise substituting hydrophobic amino acids with hydrophilicones (U.S. Pat. No. 8,637,452 and WO2015/148820 (Zhang et al.) and Zhanget al., 2018, QTY code enables design of detergent-free chemokinereceptors that retain ligand-binding activities. Proc Natl Acad Sci USA,115(37), E8652-E8659). Hydrophobic amino acids Leu, Val, Ile and Phe areexchanged by hydrophilic Gln, Thr and Tyr in the transmembrane regionsof a receptor, based on the structural and electron density mapssimilarity in their side chains. The QTY code has provided flexibilityin studying the physiological and functional properties of GPCRs, aswell as promoting their utilization, without the requirements of timeconsuming and expensive detergent screening or use of nanodisks.

The applicants reported the QTY code design of cytokine receptorscomprising an Fc domain, QTY transmembrane regions and extracellulardomains of chemokine receptors, including CCR9 and CXCR2. These QTY codedesigned GPCRs show ligand-binding properties similar to theircounterpart native receptors without the presence of hydrophobicpatches. The exemplified receptors were fused with Fc domain of mouseIgG2a protein to form an antibody-like structure. These Fc-fusionreceptors were expressed and purified in an E. coli system withsufficient yield (˜mg/L) in LB media. We also showed that the bindingaffinity of these QTY receptors approximated isolated native receptorson solution-based assays. These QTY code design of functional,water-soluble Fc-fusion GPCR proteins can be used clinically as decoytherapy to rapidly remove excessive cytokines in the setting ofhyperactive immune reactions during CRS or “cytokine storm”. Inaddition, the proteins of the invention can be used ex vivo orextracorporeally to remove cytokines from human blood. The presentinvention includes a method for the reduction in cytokine levels throughthe use of a new sorbent technology. The sorbent material can be coatedwith water soluble GPCRs directed to specific signaling molecules (e.g.,cytokines such as interferon, interleukin, chemokines) with or withoutthe use of S-layer proteins for anchoring the GPCRs. Optionally, thesorbent material can be in a cartridge and can have a variety of forms,including for example, polymer beads, glass beads, magnetic beads,porous polymers, and membranes. The blood, characterized by reducedcytokine and/or chemokine levels, can optionally be administered to thepatient. The bound cytokines and/or chemokines can be detected on thesorbent, thereby being useful as a diagnostic. The bound cytokinesand/or chemokines can optionally be recovered from the sorbent.

The invention includes QTY Fc Fusion proteins comprising an Fc domain orfragment thereof, a QTY GPCR. For example, the Fc fusion proteins of theinvention can have the formula:

N-terminus(QTY GPCR-Fc-Domain) C-terminus

Each domain or region can be directly or indirectly linked or fused toits adjacent domain. For example, additional peptide linkers (one ormore glycine residues or restriction sites) can be used to link domains.Additional domains (such as an immunoglobulin hinge region, restrictionsites, or tags) can also be used.

The invention also includes pharmaceutical compositions comprising Fcfusion QTY GPCR proteins, methods of manufacture and methods of use.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIGS. 1A and 1B illustrate the QTY strategy wherein the amino acids Q, Tand Y, together with other amino acids, can form alpha helical domainsthat mimic a transmembrane region. (A) Crystallographic electronicdensity maps of the following amino acids: Leucine (L), Asparagine (N),Glutamine (Q), Isoleucine (I), Valine (V), Threonine (T), Phenylalanine(F) and Tyrosine (Y). The density maps of L, N and Q are very similar.Likewise, the density maps of I, V and T are similar, and the densitymaps of F and Y are similar. (B) Helical wheels before and afterapplying the QTY code to transmembrane helical segment 1 (TM1) of CXCR2.Amino acids that interact with water molecules are light blue in color.The QTY code conversions render the alpha-helical segment water-soluble.

FIGS. 2A and 2B. Schematic illustration for Fc fused QTY variantcytokine receptors with antibody-like structure. (A) CXCR2^(QTY)-Fc and(B) CCR9^(QTY)-Fc. These illustrations are not to scale and thereceptors parts are significantly emphasized for clarity.

FIGS. 3A and 3B. MST ligand binding measurements. The receptors werelabeled with fluorescent dye. The ligands were purchased commerciallyfrom and dissolved in di water. (A) CXCR2^(QTY)-Fc with IL8 and (B)CCR9^(QTY)-Fc with CCL25.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

QTY Transmembrane Domain

The invention encompasses a modified, synthetic, and/or non-naturallyoccurring, α-helical domain(s) and water-soluble polypeptide (e.g.,“GPCR^(QTY)”) comprising such modified α-helical domain(s), wherein themodified α-helical domain(s) comprise an amino acid sequence in which aplurality of hydrophobic amino acid residues (L, I V, F) within aα-helical domain of a native membrane protein are replaced withhydrophilic, non-ionic amino acid residues (Q, T, T, Y, respectively, or“Q, T, Y”) and/or N and S. The invention also encompasses a method ofpreparing a water-soluble polypeptide comprising replacing a pluralityof hydrophobic amino acid residues (L, I V, F) within the α-helicaldomain(s) of a native membrane protein with hydrophilic, non-ionic aminoacid residues (Q, T, Y). The invention additionally encompasses apolypeptide prepared by replacing a plurality of hydrophobic amino acidresidues (L, I V, F) within the α-helical domain of a native membraneprotein with hydrophilic, non-ionic amino acid residues (Q, T, Y.,respectively). The variant can be characterized by the name of theparent or native protein (e.g., CXCR2) followed by the abbreviation“QTY” (e.g., CXCR2-QTY).

The present invention is directed to a method of designing, selectingand/or producing Fc fusion QTY GCPR proteins, these proteins produced bythe process, compositions comprising said polypeptides, and methods ofuse thereof. In particular, the method relates to a process fordesigning proteins using the “QTY code Principle,” changing thewater-insoluble amino acids (Leu, Ile, Val and Phe, or the single lettercode L, I, V, F) into water-soluble, non-ionic amino acids (Gln, Thr andTyr, or the single letter code Q, T, Y). Furthermore, two additionalnon-ionic amino acids Asn (N) and Ser (S) may also be used for thesubstitution for L, I and V but not for F. In the embodiments discussedbelow, it is to be understood that Asn (N) and Ser (S) are envisioned asbeing substitutable for Q and T (as a variant is described) or L, I or V(as a native protein is described). For the purposes of brevity,however, the application does not explicitly state these alternativeembodiments.

The invention utilizes a modified, synthetic, and/or non-naturallyoccurring, α-helical domain(s) and water-soluble polypeptides mimickingtransmembrane regions (e.g., “sTMs” or “QTY TMs”) comprising suchmodified α-helical domain(s), wherein the modified α-helical domain(s)comprise an amino acid sequence in which a plurality of hydrophobicamino acid residues (L, I V, F) within a α-helical domain of a nativemembrane protein are replaced with hydrophilic, non-ionic amino acidresidues (Q, T, T, Y, respectively, or “Q, T, Y”) and/or N and S. Theinvention also encompasses a method of preparing a water-solublepolypeptide comprising replacing a plurality of hydrophobic amino acidresidues (L, I V, F) within the α-helical domain(s) of a native membraneprotein with hydrophilic, non-ionic amino acid residues (Q, T, Y). Theinvention additionally encompasses a polypeptide prepared by replacing aplurality of hydrophobic amino acid residues (L, I V, F) within theα-helical domain of a native membrane protein with hydrophilic,non-ionic amino acid residues (Q, T, Y., respectively). The variant canbe characterized by the name of the parent or native protein (e.g., theCCR) preceded or followed by the abbreviation “QTY” (e.g., QTY CCR9,CCR9 QTY or CCR9^(QTY)).

In yet an additional embodiment, the native membrane protein or membraneprotein is an integral membrane protein. In a further aspect, the nativemembrane protein is a mammalian protein. The proteins of the inventionare preferably human. For the purposes of being concise, references tospecific GPCR proteins (e.g., CXCR2) are intended to refer to bothmammalian, generally, and, in the alternative, human, specifically. Inother embodiments, the α-helical domain is one of 7-transmembraneα-helical domains in a G-protein coupled receptor (GPCR) variantmodified, for example, in the extracellular or intracellular loops toimprove or alter ligand binding, as described elsewhere in theliterature. For the purposes of this invention, the word “native” isintended to refer to the protein (or α-helical domain) prior to watersolubilization in accordance with the methods described herein.

GPCRs typically have 7-transmembrane alpha-helices (7TM) and 8 non-TM.These transmembrane segments are called TM1, TM2, TM3, TM4, TM5, TM6 andTM7. The 8 non-transmembrane loops are divided into 4 extracellularloops N-terminus, EC1, EC2, EC3, and 4 intracellular non-TM, IC1, IC2,IC3, C-terminus, thus total 8 non-TMs. We can therefore divide a GPCRprotein into 15 fragments based on the transmembrane andnon-transmembrane features.

The hydrophilic residues (which replace one or more hydrophobic residuesin the α-helical domain of a native membrane protein) are selected fromthe group consisting of glutamine (Q), threonine (T), tyrosine (Y) andany combination thereof. In additional aspects, the hydrophobic residuesselected from leucine (L), isoleucine (I), valine (V) and phenylalanine(F) are replaced. Specifically, the phenylalanine residues of theα-helical domain of the protein are replaced with tyrosine; theisoleucine and/or valine residues of the α-helical domain of the proteinare replaced with threonine; and/or the leucine residues of theα-helical domain of the protein are replaced with glutamine.

The invention contemplates water soluble GPCR variants (“sGPCRs”)characterized by a plurality of transmembrane domains independentlycharacterized by at least 50%, preferably at least about 60%, morepreferably at least about 70% or 80%, such as at least about 90%) of thehydrophobic amino acid residues (I/L, V and F) of a native transmembraneprotein (e.g., GPCR) substituted by a T, Q or Y, respectively). ThesGPCRs of the invention are characterized by water solubility and ligandbinding. In particular, the sGPCR binds the same natural ligand as thecorresponding native GPCR.

CCR-9 C-C chemokine receptor type 9 isoform B: Replacing all orsubstantially all of the hydrophobic amino acids, L, I V, and F, with Q,T and Y (respectively) within the transmembrane domains results in thefollowing sequence (lower line SEQ ID NO: 1), aligned with the wild type(top line SEQ ID NO: 2):

  1 MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHFLPPLYWLVFI|||||||||||||||||||||||||||||||||||||||||||||||||..||.||....MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHYQPPQYWQTYT  61VGALGNSLVILVYWYCTRVKTMTDMFLLNLAIADLLFLVTLPFWAIAAADQWKFQTFMCK.||.|||.....|||||||||||||...|.|.||.....|.|.||.||||||||||||||TGAQGNSQTTQTYWYCTRVKTMTDMYQQNQATADQQYQTTQPYWATAAADQWKFQTFMCK 121VVNSMYKMNFYSCVLLIMCISVDRYIAIAQAMRAHTWREKRLLYSKMVCFTIWVLAAALC..|||||||.|||....||.|.|||.|.|||||||||||||..||||.|.|.|..|||.|TTNSMYKMNYYSCTQQTMCTSTDRYTATAQAMRAHTWREKRQQYSKMTCYTTWTQAAAQC 181IPEILYSQIKEESGIAICTMVYPSDESTKLKSAVLTLKVILGFFLPFVVMACCYTIIIHT.||..||||||||||||||||||||||||.|||..|.|...|...|...||||||...||TPETQYSQIKEESGIAICTMVYPSDESTKQKSATQTQKTTQGYYQPYTTMACCYTTTTHT 241LIQAKKSSKHKALKVTITVLTVFVLSQFPYNCILLVQTIDAYAMFISNCAVSTNIDICFQ..||||||||||.|.|.|..|....||.||||....||.|||||||||||.|||.|.|.|QTQAKKSSKHKAQKTTTTTQTTYTQSQYPYNCTQQTQTTDAYAMFISNCATSTNTDTCYQ 301VTQTIAFFHSCLNPVLYVFVGERFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLL.|||.|..|||.||..|...||||||||||||||||||||||||||||||||||||||||TTQTTAYYHSCQNPTQYTYTGERFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLL 361ETTSGALSL ||||||||| ETTSGALSL

Each of the predicted transmembrane regions has been underlined andexemplified a fully modified domain of the invention. Thus, for example,the invention includes a transmembrane domain comprising each underlineddomain. Preferably the protein comprising TM1 herein includes one ormore (e.g., all) of the extracellular and intracellular loop sequences(the sequences which have not been underlined). In addition oralternatively, the protein comprising the TM1 herein includes one ormore additional transmembrane regions (the underlined sequences) in thedepicted protein or homologous sequences retaining one, two, three or,possibly four or more of the native L, I V, and F amino acids, as setforth in the wild type sequence.

Coding sequences can be designed, shuffled and proteins expressed. Theexpressed proteins can be assayed for ligand binding, as describedherein.

CXCR2 chemokine receptor type 2: Replacing all or substantially all ofthe hydrophobic amino acids, L, I V, and F, with Q, T and Y(respectively) within the transmembrane domains results in the followingsequence (lower line SEQ ID NO: 3), aligned with the wild type (top lineSEQ ID NO: 4):

  1 MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHFLPPLYWLVFI|||||||||||||||||||||||||||||||||||||||||||||||||..||.||....MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHYQPPQYWQTYT  61VGALGNSLVILVYWYCTRVKTMTDMFLLNLAIADLLFLVTLPFWAIAAADQWKFQTFMCK.||.|||.....|||||||||||||...|.|.||.....|.|.||.||||||||||||||TGAQGNSQTTQTYWYCTRVKTMTDMYQQNQATADQQYQTTQPYWATAAADQWKFQTFMCK 121VVNSMYKMNFYSCVLLIMCISVDRYIAIAQAMRAHTWREKRLLYSKMVCFTIWVLAAALC..|||||||.|||....||.|.|||.|.|||||||||||||..||||.|.|.|..|||.|TTNSMYKMNYYSCTQQTMCTSTDRYTATAQAMRAHTWREKRQQYSKMTCYTTWTQAAAQC 181IPEILYSQIKEESGIAICTMVYPSDESTKLKSAVLTLKVILGFFLPFVVMACCYTIIIHT.||..||||||||||||||||||||||||.|||..|.|...|...|...||||||...||TPETQYSQIKEESGIAICTMVYPSDESTKQKSATQTQKTTQGYYQPYTTMACCYTTTTHT 241LIQAKKSSKHKALKVTITVLTVFVLSQFPYNCILLVQTIDAYAMFISNCAVSTNIDICFQ..||||||||||.|.|.|..|....||.||||....||.|||||||||||.|||.|.|.|QTQAKKSSKHKAQKTTTTTQTTYTQSQYPYNCTQQTQTTDAYAMFISNCATSTNTDTCYQ 301VTQTIAFFHSCLNPVLYVFVGERFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLL.|||.|..|||.||..|...||||||||||||||||||||||||||||||||||||||||TTQTTAYYHSCQNPTQYTYTGERFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLL 361ETTSGALSL ||||||||| ETTSGALSL

Each of the predicted transmembrane regions has been underlined andexemplified a fully modified domain of the invention. Thus, for example,the invention includes a transmembrane domain comprising each underlineddomain. Preferably the protein comprising TM1 herein includes one ormore (e.g., all) of the extracellular and intracellular loop sequences(the sequences which have not been underlined). In addition oralternatively, the protein comprising the TM1 herein includes one ormore additional transmembrane regions (the underlined sequences) in thedepicted protein or homologous sequences retaining one, two, three or,possibly four or more of the native L, I V and F amino acids, as setforth in the wild type sequence.

Coding sequences can be designed, shuffled and proteins expressed. Theexpressed proteins can be assayed for ligand binding, as describedherein.

Each QTY TM Domain can be a complete transmembrane domain or a portionthereof. For example, the QTY TM Domain can be the first 1, 2, 3, 4, ormore helical turns (approximately 4 amino acids per helical turn) fromthe extracellular surface of the transmembrane region. Additionally,amino acids that are proximal and/or external to the transmembraneregion can be included. For example, the native amino acids of theparent protein that are 1, 2, 3, 4, or 5 amino acids upstream and/ordownstream of the transmembrane region can be included in the QTY TMDomain. Optimizing the amino acids that couple or fuse the ligandbinding domain to the QTY TM Domain can control the presentation of theligand binding domain faces to the desired ligand. For example, whereligand binding domains (e.g., an alpha and beta subunit) are oriented topresent an inward face (the surface of a first ligand binding domainthat is proximal to the surface of a second ligand binding domain) whichincreases affinity or specificity for a ligand, amino acids coupling theligand binding domain to the QTY TM domain can preserve thethree-dimensional presentation of the ligand binding domain(s).

The QTY TM Domain can include one or more amino acid variants thatpresent an additional reactive moiety for functionalization. Forexample, a hydroxyl, carboxyl or amino group on an amino acid side chaincan be further functionalized, for example with a polyethylene glycol orcarbohydrate. Further functionalization can improve serum half-life ofthe Fc fusion receptor protein.

The ligand binding or extracellular domains of the GPCRs can be a nativeor wild type protein or naturally occurring alleles and splice variants,such as the extracellular domains of the receptors described herein.Where two or more ligand binding domains are required for binding, orselective binding, to the desirable ligand, that the natively outwardfaces of the ligand binding domains be preserved in selecting the linkermoieties.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt alpha-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above. QTYsubstitutions are clearly “non-conservative substitutions”, thesubstitutions are from hydrophobic to hydrophilic without introducingany charges.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine.beta.-alanine, GABA and delta-Aminolevulinic acid, 4-aminobenzoic acid(PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid,alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyricacid, gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-aminoisobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteicacid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,beta-alanine, fluoro-amino acids, designer amino acids such as betamethyl amino acids, C alpha-methyl amino acids, N alpha-methyl aminoacids, and amino acid analogs in general).

Mutations may also be made to the nucleotide sequences of theheterodimeric proteins by reference to the genetic code, includingtaking into account codon degeneracy.

In various embodiments, the present heterodimeric proteins may comprisesvariants of any of the known extracellular domains, for instance, asequence having at least about 60%, or at least about 61%, or at leastabout 62%, or at least about 63%, or at least about 64%, or at leastabout 65%, or at least about 66%, or at least about 67%, or at leastabout 68%, or at least about 69%, or at least about 70%, or at leastabout 71%, or at least about 72%, or at least about 73%, or at leastabout 74%, or at least about 75%, or at least about 76%, or at leastabout 77%, or at least about 78%, or at least about 79%, or at leastabout 80%, or at least about 81%, or at least about 82%, or at leastabout 83%, or at least about 84%, or at least about 85%, or at leastabout 86%, or at least about 87%, or at least about 88%, or at leastabout 89%, or at least about 90%, or at least about 91%, or at leastabout 92%, or at least about 93%, or at least about 94%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99%) sequence identity with the known aminoacid or nucleic acid sequences. In embodiments, a ligand binding domaincan differ from a wild type sequence, or parent protein, by 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more amino acid substitutions.

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. and has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

Fc Domain

The Fc domain of the fusion protein comprises at least a portion of aconstant immunoglobulin domain, e.g., a constant heavy immunoglobulindomain or a constant light immunoglobulin domain. Preferably, the seconddomain comprises at least a portion of a constant heavy immunoglobulindomain. The constant heavy immunoglobulin domain is preferably an Fcfragment comprising the CH2 and CH3 domain and, optionally, at least apart of the hinge region. The immunoglobulin domain may be an IgG, IgD,IgE, IgA, or IgM, immunoglobulin domain or a modified immunoglobulindomain derived therefrom. Preferably, the second domain comprises atleast a portion of a constant IgG immunoglobulin domain. The IgGimmunoglobulin domain may be selected from IgG1, IgG2, IgG3 of IgG4domains or from modified domains such as are described in U.S. Pat. No.5,925,734.

The Fc Domain is preferably a mammalian sequence, more preferably ahuman sequence, or a variant of a human sequence. The examples providedbelow illustrate a murine IgG2 Fc domain for study purposes.

Fc domain variants have been described. For example, the Fc domain cancontain one or more amino acid substitutions at amino acid residue 250,252, 254, 256, 308, 309, 311, 428, 433 or 434 (in accordance with Kabatnumbering), or equivalents thereof. For example, the amino acidsubstitution at amino acid residue 250 is a substitution with glutamine;the amino acid substitution at amino acid residue 252 is a substitutionwith tyrosine, phenylalanine, tryptophan or threonine; the amino acidsubstitution at amino acid residue 254 is a substitution with threonine;the amino acid substitution at amino acid residue 256 is a substitutionwith serine, arginine, glutamine, glutamic acid, aspartic acid, orthreonine; the amino acid substitution at amino acid residue 308 is asubstitution with threonine; the amino acid substitution at amino acidresidue 309 is a substitution with proline; the amino acid substitutionat amino acid residue 311 is a substitution with serine; the amino acidsubstitution at amino acid residue 385 is a substitution with arginine,aspartic acid, serine, threonine, histidine, lysine, alanine or glycine;the amino acid substitution at amino acid residue 386 is a substitutionwith threonine, proline, aspartic acid, serine, lysine, arginine,isoleucine, or methionine; the amino acid substitution at amino acidresidue 387 is a substitution with arginine, proline, histidine, serine,threonine, or alanine; the amino acid substitution at amino acid residue389 is a substitution with proline, serine or asparagine; the amino acidsubstitution at amino acid residue 428 is a substitution with leucine;the amino acid substitution at amino acid residue 433 is a substitutionwith arginine, serine, isoleucine, proline, or glutamine; and the aminoacid substitution at amino acid residue 434 is a substitution withhistidine, phenylalanine, or tyrosine.

In some embodiments, the Fc domain (e.g., comprising an IgG constantregion) comprises one or more mutations such as substitutions at aminoacid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabatnumbering). For example, the IgG constant region includes a tripleM252Y/S254T/T256E mutation or YTE mutation; the IgG constant regionincludes a triple H433K/N434FN436H mutation or KFH mutation; or the IgGconstant region includes an YTE and KFH mutation in combination.

In some embodiments, illustrative mutations include T250Q, M428L, T307A,E380A, 1253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A andcombinations thereof. Additional exemplary mutations in the IgG constantregion are described, for example, in Robbie, et al., AntimicrobialAgents and Chemotherapy (2013), 57(12):6147-6153, Dall'Acqua et al., JBC(2006), 281(33):23514-24, Dall'Acqua et al., Journal of Immunology(2002), 169:5171-80, Ko et al. Nature (2014) 514:642-645, Grevys et al.Journal of Immunology. (2015), 194(11):5497-508, and U.S. Pat. No.7,083,784.

The immunoglobulin domain may exhibit effector functions, particularlyeffector functions selected from ADCC and/or CDC. In some embodiments,however, modified immunoglobulin domains having modified, e.g., at leastpartially deleted, effector functions can be used.

The Fc Domain can be modified. For example, glycosylation variants canimprove or serum half. Enhanced Fc fusion protein comprising animmunoglobulin Fc region or domain comprising at least oneoligosaccharide can be produced by exposing the Fc fusion protein to atleast one glycosyltransferase. Pegylating the Fc domain can also improvethe serum half-life.

The Fc Domain can be linked to the QTY GPCR with a hinge region, derivedfrom an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive ofsubclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). Thehinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts asa flexible spacer, allowing the Fab portion to move freely in space. Incontrast to the constant regions, the hinge domains are structurallydiverse, varying in both sequence and length among immunoglobulinclasses and subclasses. For example, the length and flexibility of thehinge region varies among the IgG subclasses. The hinge region of IgG1encompasses amino acids 216-231 and, because it is freely flexible, theFab fragments can rotate about their axes of symmetry and move within asphere centered at the first of two inter-heavy chain disulfide bridges.IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and fourdisulfide bridges. The hinge region of IgG2 lacks a glycine residue, isrelatively short, and contains a rigid poly-proline double helix,stabilized by extra inter-heavy chain disulfide bridges. Theseproperties restrict the flexibility of the IgG2 molecule. IgG3 differsfrom the other subclasses by its unique extended hinge region (aboutfour times as long as the IgG1 hinge), containing 62 amino acids(including 21 prolines and 11 cysteines), forming an inflexiblepoly-proline double helix. In IgG3, the Fab fragments are relatively faraway from the Fc fragment, giving the molecule a greater flexibility.The elongated hinge in IgG3 is also responsible for its higher molecularweight compared to the other subclasses. The hinge region of IgG4 isshorter than that of IgG1 and its flexibility is intermediate betweenthat of IgG1 and IgG2. The flexibility of the hinge regions reportedlydecreases in the order IgG3>IgG1>IgG4>IgG2. In other embodiments, thelinker may be derived from human IgG4 and contain one or more mutationsto enhance dimerization (including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of CH1 to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the CH2 domain and includes residues in CH2.The core hinge region of wild-type human IgG1 contains the sequenceCys-Pro-Pro-Cys which, when dimerized by disulfide bond formation,results in a cyclic octapeptide believed to act as a pivot, thusconferring flexibility. In various embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In various embodiments, the linker of thepresent invention comprises one or more glycosylation sites.

In various embodiments, the Fc Domain may comprises variants of knowndomains, for instance, a sequence having at least about 60%, or at leastabout 61%, or at least about 62%, or at least about 63%, or at leastabout 64%, or at least about 65%, or at least about 66%, or at leastabout 67%, or at least about 68%, or at least about 69%, or at leastabout 70%, or at least about 71%, or at least about 72%, or at leastabout 73%, or at least about 74%, or at least about 75%, or at leastabout 76%, or at least about 77%, or at least about 78%, or at leastabout 79%, or at least about 80%, or at least about 81%, or at leastabout 82%, or at least about 83%, or at least about 84%, or at leastabout 85%, or at least about 86%, or at least about 87%, or at leastabout 88%, or at least about 89%, or at least about 90%, or at leastabout 91%, or at least about 92%, or at least about 93%, or at leastabout 94%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99%) sequenceidentity with the known amino acid or nucleic acid sequences. Inembodiments, the Fc Domain can differ from a wild type sequence, orparent protein, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions.

Fc QTY GPCR Proteins

Typically, QTY GPCR domain can be directly or indirectly bound to the FcDomain via an intracellular region (either the N terminal or C terminal)of the QTY GPCR. Additionally or alternatively, flexible simple aminoacid linkers (e.g., polyglycine linkers) or rigid linkers (e.g., alphahelical domain, such as a QTY TM domain), can be used.

Vectors & Polynucleotides

In other embodiments, the application provides nucleic acids encodingany of the various Fc fusion proteins disclosed herein. Codon usage maybe selected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21,2003); Sinclair et al., Protein Expr. Purif, 26(I):96-105 (October2002); Connell, N. D., Curr. Opin. Biotechnol., 12(5):446-449 (October2001); Makrides et al., Microbiol Rev., 60(3):512-538 (September 1996);and Sharp et at., Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), orAusubel, F. et at., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates,herein incorporated by reference. Generally, the DNA encoding thepolypeptide is operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, viral, orinsect genes. Such regulatory elements include a transcriptionalpromoter, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites, and sequencesthat control the termination of transcription and translation. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants is additionally incorporated.

The Fc fusion receptor proteins may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. An N-terminal leader sequence can beremoved by the host cell following expression.

For prokaryotic host cells that do not recognize and process a nativesignal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces alpha-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in U.S. Pat. No. 5,631,144. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available. The DNA for suchprecursor regions may be ligated in reading frame to DNA encoding theprotein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2-micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein disclosed herein, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tan promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein disclosed herein. Promoter sequences are known foreukaryotes. Virtually all eukaryotic genes have an AT-rich regionlocated approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tall to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding proteins disclosed herein by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the peptide-encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of mRNA encoding the protein disclosed herein.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, Rho tag, Strep tag or a GST tag. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts can be found in Cloning Vectors: A Laboratory Manual,(Elsevier, New York (1985)), the relevant disclosure of which is herebyincorporated by reference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative orgram-positive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

Receptor Protein Production

Host cells containing vectors encoding the Fc fusion receptor proteinsdescribed herein, as well as methods for producing the Fc fusionreceptor proteins are described herein. Host cells may be transformedwith the herein-described expression or cloning vectors for proteinproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Host cells usefulfor high-throughput protein production (HTPP) and mid-scale productioninclude the HMS174-bacterial strain. The host cells used to produce theproteins disclosed herein may be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma)), RPM1-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma)) are suitable for culturing thehost cells. In addition, many of the media described in Ham et al.,Meth. Enzymol., 58:44 (1979), Barites et al., Anal. Biochem., 102:255(1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655,5,122,469, 6,048,728, 5,672,502, or U.S. Pat. No. RE 30,985 may be usedas culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas Gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression and will be apparent to the ordinarilyskilled artisan.

The Fc fusion receptor proteins provided herein can also be producedusing cell-free translation systems. For such purposes the nucleic acidsencoding the fusion protein must be modified to allow in vitrotranscription to produce mRNA and to allow cell-free translation of themRNA in the particular cell-free system being utilized (eukaryotic suchas a mammalian or yeast cell-free translation system or prokaryotic suchas a bacterial ceil-free translation system.

The Fc fusion receptor proteins disclosed herein can also be produced bychemical synthesis (e.g., by the methods described in Solid PhasePeptide Synthesis, 2nd Edition, The Pierce Chemical Co., Rockford, Ill.(1984)). Modifications to the Fc fusion receptor proteins can also beproduced by chemical synthesis.

The Fc fusion receptor proteins disclosed herein can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, getfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, counter-currant distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified Fc fusion proteins are preferably at least 85% pure, orpreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the Fc fusionprotein is sufficiently pure for use as a pharmaceutical product.

QTY Design

In some aspects, the invention is directed to the use of the QTY(Glutamine, threonine and tyrosine) replacement (or “Code”) method (or“principle”) to change the transmembrane α-helix hydrophobic residuesleucine (L), isoleucine (I), valine (V), and phenylalanine (F) of anative protein to the hydrophilic residues glutamine (Q), threonine (T)and tyrosine (Y), or alternatively, as described above, Asn (N) and Ser(S) for L, I and/or V. This invention can convert a water insoluble,native membrane protein to a water-soluble counterpart.

The applicants specifically designed the QTY receptor variants to fusewith the Fc region of IgG protein in order to acquire an antibody-likestructure, as well as to promote their antibody-like functions andproperties for specific ligand recognition and binding while retainingtheir receptor characteristics. A spacer was introduced to optimize theconformation of QTY code designed receptors in the heavy chain. We usedthe Fc region of mouse IgG2a in the specific design as it is thefunctional equivalent of human IgG1. Mouse IgG is chosen over human IgGdue to the consideration of implementing mouse cytokine storm model insubsequent experiments, which is beyond the scope of current study. TheFc region can be easily exchanged in future designs. The structuralillustrations were obtained through Uniprot where applicable or from ahomology model (CXCR2) (Kwon, 2010).

Bioinformatics Analysis

QTY variant protein sequences were analyzed using a web-based tool TMHMMServer v2.0 to predict the existence of hydrophobic transmembranesegments. The server is based on a Hidden Markov Model (HMM) that takesinto account actual biological architectures of a transmembrane helixwhereas likelihood of presence is calculated (Sonnhammer et al., 1998).

Protein hydrophobicity can be plotted versus the protein sequences. TheX-axis can show the number of amino acids in sequences from N-terminusto C-terminus. In interleukin and interferon receptors, a single highprobability hydrophobic segment near the C-terminus end of each receptorand are also eliminated though QTY modification. The hydrophobicity ofboth extracellular and intracellular components is unchanged. E. coliexpression and gel-electrophoresis of QTY variant receptors. Thecorresponding genes with E. coli specific codons were synthesized andexpressed in sufficient quantities. The throughput for each receptordiffered but was all in mg/L range in LB media. All Fc fusion receptorswere expressed into inclusion bodies. They were purified by a) affinitypurification, and b) gel filtration in denatured state and then foldedinto functional state for subsequent analysis. Both arginine and DTTwere beneficial for solubilizing the proteins so either or both of themwere included in the storage buffer or for ligand binding tests.

Bold denotes transmembrane region of a receptor, within which, QTYsubstitution has occurred. Underlined parts are tags and restrictionsites used for purification and future swapping of the Fc region fortests in respective animals. Italicized sequence corresponds to the Fcregion of IgG protein, where in this case, is mouse IgG2a-Fc, thefunctional equivalent of human IgG1. The hinge region has the sequencePRGPTIKPCPPCKCPAPNLLGGP (SEQ ID NO. 5), following the underlinedsequences.

CCR9-QTY Fc (SEQ ID NO. 6)MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHYQPPQYWQTYTTGAQGNSQTTQTYWYCTRVKTMTDMYQQNQATADQQYQTTQPYWATAAADQWKFQTFMCKTTNSMYKMNYYSCTQQTMCTSTDRYTATAQAMRAHTWREKRQQYSKMTCYTTWTQAAAQCTPETQYSQIKEESGIAICTMVYPSDESTKQKSATQTQKTTQGYYQPYTTMACCYTTTTHTQTQAKKSSKHKAQKTTTTTQTTYTQSQYPYNCTQQTQTTDAYAMFISNCATSTNTDTCYQTTQTTAYYHSCQNPTQYTYTGERFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLLETTSGALSLTETSQVAPAHHHHHHHHHHTETS QVAPAHHHHHHHHHHGSKLVDPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK CXCR2-QTY Fc:(SEQ ID NO. 7) MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYYTTTTYAQTYQQSQQGNSQTMQTTQYSRVGRSVTDTYQQNQAQADQQYAQTQPTWAASKVNGWIFGTFLCKTTSQQKETNYYSGTQQQACTSTDRYLAIVHATRTLTQKRYLVKYTCQSTWGQSQQQAQPTQQYRRTVYSSNVSPACYEDMGNNTANWRMQQRTQPQSYGYTQPQQTMQYCYGFTLRTLFKAHMGQKHRAMRTTYATTQTYQQCWQPYNQTQQADTLMRTQVIQETCERRNHIDRAQDATETQGTQHSCQNPQTYAYTGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTLTETSQVAPAHHHHHHHHHHGSKLVD PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Ligand-Binding Measurement in Buffer

The affinity of QTY modified cytokine receptor fused with Fc of IgG fortheir respective native ligands was measured using MicroScaleThermophoresis (MST). Changes in thermophoretic movement for labeledproteins upon ligand-binding were recorded and plotted as a function ofligand concentration. Both QTY code designed Fc-fusion interleukin andinterferon receptors showed no non-specific adhesion or aggregationduring the measurement.

QTY Fc-fusion receptors exhibit affinity for their respective ligandstypically in nM to tens of nM range (Table 1). The affinities are lowercompared to the native receptors without Fc-fusion. For affinities ofinterleukin and interferon receptors, previous reported studiesprimarily used human neutrophils cell-based assay with isotope¹²⁵I-labeled ligand that is significantly more sensitive than using thepurified receptors measured by biophysical instrument, thus they may notbe directly comparable. The affinity K_(d) derived from MST displayssimilar values compared to previous SPR measurement on purified proteinswith the exception of IL4R^(QTY)-Fc. The method that was used todetermine the K_(d) in literature was also included in the Table.

TABLE Ligand-binding affinity of Fc fused QTY cytokine receptors Native(K_(d), nM) QTY variant (K_(d), nM) CCR9^(QTY)-Fc vs CCL25 ~8 37.2 ±15.7 (Eberhardson et al., 2017) CXCR2^(QTY)-Fc vs IL8 0.5 ± 0.3(Monomer) 60.3 ± 21.0 8.5 ± 2.0 (Dimer) (Rajarathnam et al., 2006)

The applicants' Fc-fusion water-soluble receptors, as a decoy, canrapidly soak up excessive cytokines during “cytokine storm” unleashedduring CAR-T treatment and COVID-19. When the designed water-solubleFc-receptors bind to excessive cytokines, they act as decoy to preventthe excessive cytokine to directly interact with target cells, thereforereducing the organ damage and toxicity conferred by “cytokine storm”.There are over 20 Fc-fusion proteins commercially available and severalof these have been developed as therapeutics (Czajkowsky et al., 2012).Although there have been many Fc-fusion proteins developed for variousapplications, they are water-soluble proteins (Czajkowsky et al., 2012;Mekhaiel et al., 2011). The applicants' QTY code designed Fc-fusionreceptors provide a novel platform for further design of other types offusion membrane receptors for therapeutic and diagnostic applications.

The design and synthesis of functionally equivalent transmembraneproteins have implications beyond biological and clinical use. Highlyspecific towards their respective ligands, QTY code modifiedtransmembrane proteins can also serve as ideal candidates for molecularsensing. Complex electrical arrays functionalized with a variety typesof water-soluble membrane proteins can potentially mimic cell responsein vitro and be fabricated into a pseudo cell with electrical readouts.

Thus, one or more Fc-fusion proteins of the invention can beadministered to a patient in need thereof, (e.g., to treat cytokinestorm) in a therapeutically effective amount to bind one or more cognatecytokines. In addition, one or more Fc-fusion proteins of the inventioncan be contacted with blood, e.g., extracorporeally, to a patient inneed thereof, (e.g., to treat cytokine storm) in a therapeuticallyeffective amount to bind one or more cognate cytokines.

Typically, extracorporeal treatment to remove cytokines from a patient'sblood stream will benefit from immobilizing the Fc fusion protein(s) ofthe invention. Thus, the invention includes a sorbent material having asubstrate (e.g., polymer beads, glass beads, magnetic beads, porouspolymers, membranes) upon which an Fc fusion protein of the invention isimmobilized. Immobilization can be accomplished by covalent ornon-covalent binding. The sorbent material can be loaded into acartridge. The sorbent material of the invention can also be used indiagnostics to screen and identify the presence (and/or quantity) of oneor more cytokines.

The Fc fusion proteins can be directly or indirectly bound to asubstrate or absorbent material. Binding with a so-called S-layer allowsdirected patterned binding. Binding without an S-layer can occur in arandom oriented fashion, leading to a less dense distribution on thesurface and the potential for “empty spaces” on the support.Non-specific adsorption of serum components may occur with randomdistribution. A functional S-layer lattice can be stabilized both byinter- and intramolecular cross-linking within the protein lattice andby introducing (cross)linkers between the S-layer and the supportinglayer. Such structures reveal a considerable higher chemical andmechanical stability compared to functional layers composed of randomlybound molecules which are only cross linked to the supporting layer.Support-structures (beads, plane surfaces, gels) functionalized withProtein A can be a useful starting material for generating products ofthe invention. Such matrices could be used for direct binding theantibody-like receptor molecules. Nevertheless, such composites will beless stable and reveal more leakage than S-layer based material evenafter crosslinking. It is anticipated that such materials are suitablefor the first in vitro studies using cytokine spiked plasma.

In one embodiment, a substrate comprising self-assembled Fc fusionproteins of the invention, as generally described in WO 2019/046699,which is incorporated herein by reference. Specifically, the N-terminusof the S-layer fusion protein binds to the solid substrate or support.Thus, the self-assembling unit bound to the surface of the substrate cancomprise elements arranged as follows:

Substrate Surface—[N-S-layer protein-C-Fusion Domain]—[(BindingMoiety-C-QTY N];

wherein “N” and “C” indicate the N and C-termini, respectively, andwherein --- represents covalent or non-covalent, direct or indirect,attachment of the S-layer fusion protein to the substrate surface andbinding of the fusion domain of the S-layer fusion protein to thebinding moiety of the fusion protein.

Substrates include organic and inorganic, hydrophobic or hydrophilicmaterials, such as polystyrene surfaces, silicon wafers (SiO2, Si3N4,hydrophilic, and/or hydrophobic), gold wafers, glass, metal oxidesurfaces (for example, aluminum oxide, indium tin oxide), stainlesssteel, modified graphene, carbon nanotubes, poly-lysine modifiedsurfaces, magnetic beads, ELISA plates, silica beads, filling materialsfor column chromatography, coating resins for blood purification, andpolyamide membranes. For example, the surface can be a semi-conductingor conducting surface, including, but not limited to, silicon, gold,conducting polymers, carbon nanotubes, and graphene. For example, thesurface protein can recrystallize on a silicon wafer, a silicondioxide-coated silicon wafer, indium tin oxide (ITO) coated glass, orTiO₂—SiO₂ hybrid sol-gel coated glass. In an additional example, thesubstrate can be surface-treated with poly-l-lysine, for example,poly-l-lysine-treated gold. In certain additional aspects, the substratecan be flexible plastic, for example, ITO coated plastic film, graphenecoated film, or TiO₂—SiO₂ hybrid sol-gel coated film. In certainadditional aspects, the solid support is a sensor chip of a surfaceplasmon resonance system.

The surface preferably comprises a high density of proteins, such asbetween about 2.37 to about 4.37×10¹² per 1 cm². In certain embodiments,at least two, three, four, five or more different cytokine receptorvariants can be immobilized on the substrate. For example, at least twodifferent receptor variants (for example, CXCR2 and CCR9 variants) canbe immobilized on the surface.

The proteins can preferably crosslinked, thereby improving stability onthe surface. Examples include dimethyl pimelimidate, glutaraldehyde,bis(sulfosuccinimidyl)superat, amino-carboxyl group directedcrosslinkers including, for example,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and other amine-aminodirected linkers. Cross linked S-layer lattices can avoid leakage, whichis preferred for extracorporeal blood purification.

Substrates with immobilized Fc fusion proteins can be loaded intocartridges, which may be arranged in series or in parallel. In sucharrangements more than one type of adsorbent (e.g., each with adifferent cytokine receptor) may be linked to the blood streamsimultaneously. The progress of cytokine removal during the procedure(concentration of cytokines in the plasma) can be simultaneouslymonitored and rapidly stopped if the desired concentration is achieved.Time-controlled procedures can offer a great advantage in case ofemergency treatment.

COVID-19 infection can be associated with severe Thrombosis. The causefor this side effect of the viral infection is obviously not yetunderstood. In comparison to treatments involving injection ofwater-soluble cytokine receptors, risk for Thrombosis can be mitigatedwith extracorporeal therapy. Extracorporeal sorbents (Apheresisprocedures) may benefit from a faster approval process.

A potential application for extracorporeal purification can bedetoxification. QTY GPCR proteins with high binding capacities forspecific drugs can have a great potential to remove the components(drugs) from the blood stream. This could open a field and stimulate thescreening for new “binding sites” (drug/protein interaction).

The invention contemplates a process for direct binding of the watersoluble (QTY code modified) GPCR proteins and derived antibody-likereceptor molecules for sorbent technologies for cytokine removal inhuman septic shock and for producing matrices for diagnostic tools(e.g., solid phase immunoassays, ELISA, QCMD).

In addition, the sorbent can be used in the purification of thecytokines and/or chemokines, such as from cell culture supernatants. Forexample, the sorbent can be used in chromatography column. Cross-linkingthe protein matrices as described above can be beneficial. The cytokinesand/or chemokines can be removed from the sorbent according to knowntechniques. Sorbents utilizing magnetic beads can facilitate isolationand recovery. The sorbents of the invention can reduce production costs.

Materials and Methods Genes Identification and QTY Modification

Sequences of the respective proteins were obtained from Uniprot:www.uniprot.org/. Respective extracellular, transmembrane andcytoplasmic domains were identified. QTY code was only applied to thetransmembrane helical domain to solubilize the proteins.

Bioinformatics Analysis

Protein properties were calculated based on their primary sequences viathe open access web-based tool ExPASy:https://web.expasy.org/protparam/. The existence of hydrophobic patchwithin the transmembrane region in native and QTY variant proteinsequences was determined via the open access web-based tool TMHMM Serverv.2.0: www.cbs.dtu.dk/services/TMHMM-2.0.

E. coli Expression System and Protein Purification

Genes of QTY modified cytokine receptor proteins were cloned into Fcregion of mouse IgG2a which is the functional equivalence of human IgG1.The full sequences were codon optimized for E. coli expression andobtained from Genscript. The genes were cloned into pET20b expressionvector with Carbenicillin resistance. The plasmids were reconstitutedand transformed into E. coli BL21(DE3) strain. Transformants wereselected on LB medium plates with 100 μg/ml Carbenicillin. E. colicultures were grown at 37° C. until the OD₆₀₀ reached 0.4-0.8, afterwhich IPTG (isopropyl-D-thiogalactoside) was added to a finalconcentration of 1 mM followed by 4-hour expression. Cells were lysed bysonication in B-PER™ protein extraction agent (Thermos-Fisher) andcentrifuged (23,000×g, 40 min, 4° C.) to collect the inclusion body. Thebiomass was then subsequently washed twice in buffer 1 (50 mM Tris.HClpH7.4, 50 mM NaCl, 10 mM CaCl2, 0.1% v/v Trition X100, 2M Urea, 0.2 μmfiltered), once in buffer 2 (50 mM Tris.HCl pH7.4, 1M NaCl, 10 mM CaCl2,0.1% v/v Trition X100, 2M Urea, 0.2 μm filtered) and again in buffer 1.Pellets from each washing step were collected by centrifugation(23,000×g, 25 min, 4° C.).

Washed inclusion bodies were fully solubilized in denaturation buffer(6M guanidine hydrochloride, 1×PBS, 10 mM DTT, 0.2 μm filtered) at roomtemperature for 1.5 hour with magnetic stirring. The solution wascentrifuged at 23,000×g for 40 min at 4° C. The supernatant withproteins was then purified by Qiagen Ni-NTA beads (His-tag) followed bysize exclusion chromatography using an ÅKTA Purifier system and a GEhealthcare Superdex 200 gel-filtration column. Purified protein wascollected and dialyzed twice against renaturation buffer (50 mM Tris.HCl pH 9.0, 3 mM reduced glutathione, 1 mM oxidized glutathione, 5 mMethylenediaminetetraacetic acid, and 0.5M L-arginine). Following anovernight refolding process, the re-natured protein solution wasdialyzed into storage buffer of 50 mM Tris. HCl pH 9.0 with variousarginine content.

Microscale Thermophoresis

MicroScale Thermophoresis (MST) is an optical method detecting changesin thermophoretic movement and TRIC of the protein-attached fluorophoreupon ligand binding. Active labelled proteins contribute to thethermophoresis signal upon ligand binding. Inactive proteins influencethe data as background but not the signals and only data from bindingproteins are used to derive the K_(d) value. Herein ligand bindingexperiments were carried out with 5 nM NT647-labeled protein in 1×PBS,10 mM DTT buffer with different concentration of arginine, against agradient of respective ligands on a Monolith NT.115 pico instrument at25° C. Synthesized receptors were labeled with Monolith NT™ 2^(nd)generation protein labeling kit RED-NHS (NanoTemper Technologies) so asto obtain unique fluorescent signals. MST time traces were recorded andanalyzed to obtain the highest possible signal-to-noise levels andamplitudes, >5 Fnorm units. The recorded fluorescence was plottedagainst the concentration of ligand, and curve fitting was performedusing the K_(d) fit formula derived from the law of mass action. Forclarity, binding graphs of each independent experiment were normalizedto the fraction bound (0=unbound, 1=bound). MST experiments wereperformed in the Center for Macromolecular Interactions at HarvardMedical School.

K_(d) Fitting Model:

K_(d) model is the standard fitting model based on law of mass action.

Curve Fit Formula:

${F\left( c_{T} \right)} = {F_{u} + {\left( {F_{b} - F_{u}} \right)*\frac{c_{AT}}{c_{A}}}}$$\frac{c_{AT}}{c_{A}} = {{{fraction}\mspace{14mu}{bound}} = {\frac{1}{2c_{A}}*\left( {c_{T} + c_{A} + K_{D} - \sqrt{\left( {c_{T} + c_{A} + K_{D}} \right)^{2} - {4c_{T}c_{A}}}} \right)}}$

F_(u): fluorescence in unbound stateF_(b): fluorescence in bound stateK_(D): dissociation constant, to be determinedc_(AT): concentration of formed complexC_(A): constant concentration of molecule A (fluorescent), knownC_(T): concentration of molecule T in serial dilution.In broad terms, the protein design process comprises all, orsubstantially all, the steps:

-   -   (1) identifying a first transmembrane region by predicting an        alpha-helical structure of a protein;    -   (2) modifying a plurality of hydrophobic amino acids via the QTY        Code, as defined herein to obtain a modified first transmembrane        sequence.

Step-by-Step Description:

1: In step 1, a computer interface of a computer system receives aprotein sequence, selected for analysis, and data descriptive of theprotein (e.g., the sequence) entered, uploaded or inputted through acomputer interface of a computer system. The data entered can be aprotein name, a database reference, or a protein sequence. For example,the protein sequence can be uploaded through a computer interface.

2: In step 2, additional data about the protein can be identified,determined, obtained and/or entered, including its name or sequence andentered via the computer interface. One source to obtain protein data isa database named UniProt (www.uniprot.org/). Alternatively, the methodof the invention can store data relating to the protein, or relatedsequences to the protein, for later retrieval by the user in this step.In embodiments, the program can prompt the user to select a database orfile for retrieving additional data (e.g., sequence data) relating tothe protein selected for analysis.

3: In step 3, the user can enter, upload, or obtain data identifying thetransmembrane regions. For example, the user can be prompted to obtainthe data from a public source, such as from UniProt. The information canbe collected from the database for use in Step 5.

4: Alternatively or additionally, the transmembrane region can bepredicted by the method. Transmembrane regions are generallycharacterized by an alpha helical conformation. Transmembrane helixprediction can be predicted using a software package named TMHMM 2.0(TransMembrane prediction using Hidden Markov Models), developed byCenter for Biological Sequence Analysis(http://www.cbs.dtu.dk/services/TMHMM/). Some versions of the softwarehave some problems on peak finding and sometimes fails to find TMregions. Therefore, in a preferred embodiment, a modified version of theprogram is used, wherein the peak searching method execute by thecomputer system introduces a dynamic baseline. Here, if the TMs usingthe initial baseline value are not found, the baseline can be changed toa lower value. For example, the default baseline is 0.2. One can set thebaseline value to 0.1. If too many TMs are found, the baseline can bechanged to a higher value, such as 0.15.

5: After identifying the TM data in the form, the sequence of a proteinis divided into fragments including transmembrane segments andnon-transmembrane segments.

It is understood that the system can execute one or more, such as all ofthe steps described above, using a computer interface for input by auser.

A first transmembrane region (typically, but not essentially, thetransmembrane region which is most proximal to the N-terminal of theprotein) is selected for variation. Hydrophobic amino acids (L, I, V,and F) are then substituted with the corresponding hydrophilic aminoacid (Q, T or Y). It is understood that the amino acid is not actuallysubstituted into the protein, in this context. Rather, the amino aciddesignation is substituted in the sequence for modeling. Thus, the term“sequence” is intended to include “sequence data.” Typically, most orall of the hydrophobic amino acids are selected for substitution. Ifless than all amino acids are selected, it may be desirable to selectthe internal hydrophobic amino acids leaving one or more N and/or Cterminal amino acids of the transmembrane regions hydrophobic.Additionally or alternatively, it may be desirable to select to replaceall of the leucines (L) in a transmembrane region. Additionally oralternatively, it may be desirable to select to replace all of theisoleucines (I) in a transmembrane region. Additionally oralternatively, it may be desirable to select to replace all of thevalines (V) in a transmembrane region. Additionally or alternatively, itmay be desirable to select to replace all of the phenylalanines (F) in atransmembrane region. Additionally or alternatively, it can bebeneficial to retain one or more phenylalanines in the transmembraneregion. Additionally or alternatively, it can be beneficial to retainone or more valines in the transmembrane region. Additionally oralternatively, it can be beneficial to retain one or more leucine in thetransmembrane region. Additionally or alternatively, it can bebeneficial to retain one or more isoleucines in the transmembraneregion. Additionally or alternatively, it can be beneficial to retainone or more hydrophobic amino acids in the transmembrane region wherethe wild type sequence is characterized by three or more contiguoushydrophobic amino acids. The transmembrane region so designed (thetransmembrane variant or “variant”) is then subjected to thetransmembrane region prediction process, as discussed herein.

The nucleic acid molecules are preferably designed to provide codonoptimization and intron deletions for the expression systems selected toproduce a library of coding sequences. For example, if the expressionsystem is E. coli, codons optimized for E. coli expression can beselected. www.dna20.com/resources/genedesigner. In addition, a promoterregion, such as a promoter suitable for expression in the expressionsystem (e.g., E. coli) is selected and operatively connected to thecoding sequences in the library of coding sequences.

The initial library of coding sequences, or a portion thereof, is thenexpressed to produce a library of proteins. The library is thensubjected to a ligand binding assay. In the binding assay, fusionproteins are contacted with the ligand, preferably in an aqueous mediumand ligand binding is detected.

The invention includes transmembrane domain variants, and nucleic acidmolecules encoding same, obtained, or obtainable, from the methodsdescribed herein.

The invention further encompasses a method of treatment for a disorderor disease that is mediated by the activity of a membrane protein,comprising the use of a water-soluble polypeptide to treat saiddisorders and diseases, wherein said water-soluble polypeptide comprisesa modified α-helical domain, and wherein said water-soluble polypeptideretains the ligand-binding activity of the native membrane protein.Examples of such disorders and diseases include, but are not limited to,cancer, small cell lung cancer, melanoma, breast cancer, Parkinson'sdisease, cardiovascular disease, hypertension, and asthma.

As described herein, the water-soluble peptides described herein can beused for the treatment of conditions or diseases mediated by theactivity of a membrane protein. In certain aspects, the water-solublepeptides can act as “decoys” for the membrane receptor and bind to theligand that otherwise activates the membrane receptor. As such, thewater-soluble peptides described herein can be used to reduce theactivity of a membrane protein. These water-soluble peptides can remainin the circulation and competitively bind to specific ligands, therebyreducing the activity of membrane bound receptors.

The invention also encompasses a pharmaceutical composition comprisingsaid water-soluble polypeptide and a pharmaceutically acceptable carrieror diluent.

The compositions can also include, depending on the formulation desired,pharmaceutically acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the pharmacologic agentor composition. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized SEPHAROSE™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes).

The compositions can be administered parenterally such as, for example,by intravenous, intramuscular, intrathecal or subcutaneous injection.Parenteral administration can be accomplished by incorporating acomposition into a solution or suspension. Such solutions or suspensionsmay also include sterile diluents such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents. Parenteral formulations may also includeantibacterial agents such as, for example, benzyl alcohol or methylparabens, antioxidants such as, for example, ascorbic acid or sodiumbisulfate and chelating agents such as EDTA. Buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose may also be added. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Additionally, auxiliary substances, such as wetting or emulsifyingagents, surfactants, pH buffering substances and the like can be presentin compositions. Other components of pharmaceutical compositions arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, and mineral oil. In general, glycols such aspropylene glycol or polyethylene glycol are preferred liquid carriers,particularly for injectable solutions.

Injectable formulations can be prepared either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide, or copolymer for enhancedadjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 andHanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The compositionsand pharmacologic agents described herein can be administered in theform of a depot injection or implant preparation which can be formulatedin such a manner as to permit a sustained or pulsatile release of theactive ingredient.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches,ointments, creams, gels, salves and the like. Transdermal delivery canbe achieved using a skin patch or using transferosomes. [Paul et al.,Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta1368: 201-15, 1998].

“Treating” or “treatment” includes preventing or delaying the onset ofthe symptoms, complications, or biochemical indicia of a disease,alleviating or ameliorating the symptoms or arresting or inhibitingfurther development of the disease, condition, or disorder. A “patient”is a human subject in need of treatment.

An “effective amount” refers to that amount of the therapeutic agentthat is sufficient to ameliorate of one or more symptoms of a disorderand/or prevent advancement of a disorder, cause regression of thedisorder and/or to achieve a desired effect.

The words “a” or “an” are meant to encompass one or more, unlessotherwise specified.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

-   BAYBUTT, T. R., FLICKINGER, J. C., CAPAROSA, E. M. & SNOOK, A. E.    (2019). Advances in chimeric antigen receptor T-Cell therapies for    solid tumors. Clinical Pharmacology & Therapeutics, 105(1), 71-78.-   CACCURI, F., GIAGULLI, C., BUGATTI, A., BENETTI, A., ALESSANDRI, G.,    RIBATTI, D., MARSICO, S., APOSTOLI, P., SLEVIN, M. A., RUSNATI, M.,    GUZMAN, C. A., FIORENTINI, S. & CARUSO, A. (2012). HIV-1 matrix    protein p17 promotes angiogenesis via chemokine receptors CXCR1 and    CXCR2. Proceedings of the Proc Natl Acad Sci USA, 109(36),    14580-14585.-   CELADA, A., ALLEN, R., ESPARZA, I., GRAY, P. W. & SCHREIBER, R. D.    (1985). Demonstration and partial characterization of the    interferon-gamma-receptor on human mononuclear phagocytes. Journal    of Clinical Investigation, 76(6), 2196-2205.-   CZAJKOWSKY, D. M., HU, J., SHAO, Z. F. & PLEASS, R. J. (2012).    Fc-fusion proteins: new developments and future perspectives. EMBO    Molecular Medicine, 4(10), 1015-1028.-   DE JONG, M. D., SIMMONS, C. P., THANH, T. T., HIEN, V. M.,    SMITH, G. J. D., CHAU, T. N. B., HOANG, D. M., CHAU, N. V. V.,    KHANH, T. H., DONG, V. C., QUI, P. T., VAN CAM, B., HA, D. Q., GUAN,    Y., PEIRIS, J. S. M., CHINH, N. T., HIEN, T. T. & FARRAR, J. (2006).    Fatal outcome of human influenza A (H5N1) is associated with high    viral load and hypercytokinemia. Nature Medicine, 12(10), 1203-1207.-   DRAZEN, J. M. C., R. L.; GOLDMAN, L.; BENNETT, C. (2000). Cecil    textbook of medicine, 21st edition edition. Philadelphia: W.B.    Saunders.-   EBERHARDSON, M., KARLEN, P., LINTON, L., JONES, P., LINDBERG, A.,    KOSTALLA, M. J., LINDH, E., ODEN, A., GLISE, H. & WINQVIST, O.    (2017). Randomised, double-blind, placebo-controlled trial of    CCR9-targeted leukapheresis treatment of ulcerative colitis    patients. Journal of Crohns & Colitis, 11(5), 534-542.-   HU, Y., ZHANG, L., WU, R. R., HAN, R. F., JIA, Y. H., JIANG, Z. M.,    CHENG, M. X., GAN, J., TAO, X. & ZHANG, Q. P. (2011). Specific    killing of CCR9 high-expressing acute T lymphocytic leukemia cells    by CCL25 fused with PE38 toxin. Leukemia Research, 35(9), 1254-1260.-   HUANG, C., WANG, Y. & LI, X. (2020). Clinical features of patients    infected with 2019 novel coronavirus in Wuhan, China (vol 395, pg    497, 2020). Lancet, 395(10223), 496-496.-   ITTERSHAGEN, S., ERICSON, S., ELDJEROU, L., SHOJAEE, A., BLEICKARDT,    E., PATEL, M., TARAN, T., ANAK, O., HALL, C., LEUNG, M.,    ROCCOBERTON, D., SALMON, F., FUCHS, M., ROMANOV, V. & LEBWOHL, D.    (2019). Industry's giant leap into cellular therapy: catalyzing    chimeric antigen receptor T cell (CAR-T) immunotherapy. Current    Hematologic Malignancy Reports, 14(1), 47-55.-   JAIN, M. D. & DAVILA, M. L. (2018). Concise Review: Emerging    principles from the clinical application of chimeric antigen    receptor t cell therapies for B cell malignancies. Stem Cells,    36(1), 36-44.-   JERABEK-WILLEMSEN, M., ANDRE, T., WANNER, R., ROTH, H. M., DUHR, S.,    BAASKE, P. & BREITSPRECHER, D. (2014). MicroScale thermophoresis:    interaction analysis and beyond. Journal of Molecular Structure,    1077, 101-113.-   KWON, H. R. (2010). Study of the structure and function of CXC    chemokine receptor 2. Master's Thesis, University of Tennessee.-   LAPORTE, S. L., JUO, Z. S., VACLAVIKOVA, J., COLF, L. A., QI, X. L.,    HELLER, N. M., KEEGAN, A. D. & GARCIA, K. C. (2008). Molecular and    structural basis of cytokine receptor pleiotropy in the    interleukin-4/13 system. Cell, 132(2), 259-272.-   MEKHAIEL, D. N. A., CZAJKOWSKY, D. M., ANDERSEN, J. T., SHI, J. G.,    EL-FAHAM, M., DOENHOFF, M., MCINTOSH, R. S., SANDLIE, I., HE, J. F.,    HU, J., SHAO, Z. F. & PLEASS, R. J. (2011). Polymeric human    Fc-fusion proteins with modified effector functions. Scientific    Reports, 1.-   MIKULECKY, P., CERNY, J., BIEDERMANNOVA, L., PETROKOVA, H., KUCHAR,    M., VONDRASEK, J., MALY, P., SEBO, P. & SCHNEIDER, B. (2013).    Increasing affinity of interferon-gamma receptor 1 to    interferon-gamma by computer-aided design. Biomed Research    International.-   QING, R., HAN, Q. Y., SKUHERSKY, M., CHUNG, H., BADR, M.,    SCHUBERT, T. & ZHANG, S. G. (2019). QTY code designed thermostable    and water-soluble chimeric chemokine receptors with tunable ligand    affinity. Proc Natl Acad Sci USA, 116(51), 25668-25676.-   RAJARATHNAM, K., PRADO, G. N., FERNANDO, H., CLARK-LEWIS, I. &    NAVARRO, J. (2006). Probing receptor binding activity of    interleukin-8 dimer using a disulfide trap. Biochemistry, 45(25),    7882-7888.-   RICHTER, D., MORAGA, I., WINKELMANN, H., BIRKHOLZ, O., WILMES, S.,    SCHULTE, M., KRAICH, M., KENNEWEG, H., BEUTEL, O., SELENSCHIK, P.,    PATEROK, D., GAVUTIS, M., SCHMIDT, T., GARCIA, K. C., MULLER, T. D.    & PIEHLER, J. (2017). Ligand-induced type II interleukin-4 receptor    dimers are sustained by rapid re-association within plasma membrane    microcompartments. Nature Communications, 8, No. 15976.-   SAVARIN, C. & BERGMANN, C. C. (2018). Fine Tuning the Cytokine Storm    by IFN and IL-10 Following Neurotropic Coronavirus    Encephalomyelitis. Frontiers in Immunology, 9, 3022.-   SHIMABUKURO-VORNHAGEN, A., GODEL, P., SUBKLEWE, M., STEMMLER, H. J.,    SCHLOSSER, H. A., SCHLAAK, M., KOCHANEK, M., BOLL, B. & VON    BERGWELT-BAILDON, M. S. (2018). Cytokine release syndrome. Journal    for Immunotherapy of Cancer, 6 (1) 56.-   SOMOVILLA-CRESPO, B., MONZON, M. T. M., VELA, M., CORRALIZA-GORJON,    I., SANTAMARIA, S., GARCIA-SANZ, J. A. & KREMER, L. (2018). 92R    monoclonal antibody inhibits human CCR9(+) leukemia cells growth in    NSG mice xenografts. Frontiers in Immunology, 9.-   SONNHAMMER, E. L., VON HEIJNE, G. & KROGH, A. (1998). A hidden    Markov model for predicting transmembrane helices in protein    sequences. In Ismb, vol. 6, pp. 175-182.-   SRIVASTAVA, S. & RIDDELL, S. R. (2015). Engineering CAR-T cells:    Design concepts. Trends in Immunology, 36(8), 494-502.-   TAN, J. C., INDELICATO, S. R., NARULA, S. K., ZAVODNY, P. J. &    CHOU, C. C. (1993). Characterization of interleukin-10 receptors on    human and mouse cells. J. Biological Chemistry, 268(28),    21053-21059.-   TISONCIK, J. R., KORTH, M. J., SIMMONS, C. P., FARRAR, J.,    MARTIN, T. R. & KATZE, M. G. (2012). Into the eye of the cytokine    storm. Microbiology and Molecular Biology Reviews, 76(1), 16-32.-   TU, Z. B., XIAO, R. J., XIONG, J., TEMBO, K. M., DENG, X. Z., XIONG,    M., LIU, P., WANG, M. & ZHANG, Q. P. (2016). CCR9 in cancer:    oncogenic role and therapeutic targeting. J. Hematology & Oncology,    9:10 doi: 10.1186/s13045-016-0236-7.-   XU, X. J. & TANG, Y. M. (2014). Cytokine release syndrome in cancer    immunotherapy with chimeric antigen receptor engineered T cells.    Cancer Letters, 343(2), 172-178.-   XU, Z., SHI, L., WANG, Y., ZHANG, J., HUANG, L., ZHANG, C., LIU, S.,    ZHAO, P., LIU, H. & ZHU, L. (2020). Pathological findings of    COVID-19 associated with acute respiratory distress syndrome. The    Lancet Respiratory Medicine. pii: S2213-2600(20)30076-X. doi:    10.1016/S2213-2600(20)30076-X.-   ZHANG, S. G., TAO, F., QING, R., TANG, H. Z., SKUHERSKY, M., CORIN,    K., TEGLER, L., WASSIE, A., WASSIE, B., KWON, Y., SUTER, B.,    ENTZIAN, C., SCHUBERT, T., YANG, G., LABAHN, J., KUBICEK, J. &    MAERTENS, B. (2018). QTY code enables design of detergent-free    chemokine receptors that retain ligand-binding activities. Proc Natl    Acad Sci USA, 115(37), E8652-E8659.

All documents and references described herein are individuallyincorporated by reference to into this document to the same extent as ifthere were written in this document in full or in part.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although many methods andreagents are similar or equivalent to those described herein, theexemplary methods and materials are disclosed herein.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A Fc fusion QTY CCR9 variant receptor proteincomprising an Fc domain and one or more QTY CCR9 variant receptordomain.
 2. The Fc fusion QTY CCR9 variant receptor protein of claim 1,wherein the QTY CCR9 variant receptor domain comprises one or more QTYtransmembrane regions characterized by a plurality of amino acidsselected from the group consisting of Q, T and Y and is an alpha helix.3. The Fc fusion QTY CCR9 variant receptor protein of claim 1, whereineach transmembrane region has the amino acid sequence of a cytokinereceptor transmembrane domain wherein a plurality of hydrophobic aminoacids is replaced with a Q, Tor Y.
 4. The Fc fusion QTY CCR9 variantreceptor protein of claim 3, wherein all or substantially all of theleucines (L) in each transmembrane domain are replaced with glutamines(Q).
 5. The Fc fusion QTY CCR9 variant receptor protein of claim 3,wherein all or substantially all of the valines (V) in eachtransmembrane domain are replaced with threonines (T).
 6. The Fc fusionrec QTY CCR9 variant receptor protein of claim 3, wherein all orsubstantially all of the isoleucines (I) in each transmembrane domainare replaced with threonines (T).
 7. The Fc fusion QTY CCR9 variantreceptor protein of claim 3, wherein all or substantially all of thephenylalanines (F) in each transmembrane domain are replaced withtyrosines (Y).
 8. The Fc fusion QTY CCR9 variant receptor protein ofclaim 5, wherein all or substantially all of the internal L, V, I and/orFs in each transmembrane domain are replaced with Q, T, T and Y,respectively.
 9. The Fc fusion QTY CCR9 variant receptor protein ofclaim 1, wherein the Fc domain comprises an IgG constant domain.
 10. TheFc fusion QTY CCR9 variant receptor protein of claim 1, wherein the FcDomain is fused to the QTY CCR9 variant receptor domain via a hingeregion.
 11. A Fc fusion QTY CXCR2 variant receptor protein comprising anFc domain and one or more QTY CXCR2 variant receptor domain.
 12. The Fcfusion QTY CXCR2 variant receptor protein of claim 1, wherein the QTYCXCR2 variant receptor domain comprises one or more QTY transmembraneregions characterized by a plurality of amino acids selected from thegroup consisting of Q, T and Y and is an alpha helix.
 13. The Fc fusionQTY CXCR2 variant receptor protein of claim 1, wherein eachtransmembrane region has the amino acid sequence of a cytokine receptortransmembrane domain wherein a plurality of hydrophobic amino acids isreplaced with a Q, Tor Y.
 14. The Fc fusion QTY CXCR2 variant receptorprotein of claim 3, wherein all or substantially all of the leucines (L)in each transmembrane domain are replaced with glutamines (Q).
 15. TheFc fusion QTY CXCR2 variant receptor protein of claim 3, wherein all orsubstantially all of the valines (V) in each transmembrane domain arereplaced with threonines (T).
 16. The Fc fusion rec QTY CXCR2 variantreceptor protein of claim 3, wherein all or substantially all of theisoleucines (I) in each transmembrane domain are replaced withthreonines (T).
 17. The Fc fusion QTY CXCR2 variant receptor protein ofclaim 3, wherein all or substantially all of the phenylalanines (F) ineach transmembrane domain are replaced with tyrosines (Y).
 18. The Fcfusion QTY CXCR2 variant receptor protein of claim 5, wherein all orsubstantially all of the internal L, V, I and/or Fs in eachtransmembrane domain are replaced with Q, T, T and Y, respectively. 19.The Fc fusion QTY CXCR2 variant receptor protein of claim 1, wherein theFc domain comprises an IgG constant domain.
 20. The Fc fusion QTY CXCR2variant receptor protein of claim 1, wherein the Fc Domain is fused tothe QTY CCR9 variant receptor domain via a hinge region.
 21. Apharmaceutical composition comprising the protein of claim 1, and apharmaceutically acceptable carrier.
 22. A nucleic acid encoding theprotein of claim
 1. 23. A method of treating a cytokine storm in apatient in need thereof comprising administering a pharmaceuticalcomposition according to claim
 21. 24. A method of treating a cancer ina patient in need thereof comprising administering a pharmaceuticalcomposition according to claim 21 in combination with CAR-Timmunotherapy.
 25. A method of reducing cytokine levels from human bloodcomprising the steps of removing human blood from a patient in needthereof and contacting the human blood with a sorbent comprising a Fcfusion QTY variant receptor of claim
 1. 26. A sorbent comprising a Fcfusion QTY variant receptor of claim 1, immobilized upon a substrate.27. A method of isolating a cytokine from a stream or sample comprisingcontacting the stream or sample with a sorbent of claim 26 underconditions suitable for binding the cytokine onto the sorbent andrecovering the cytokine.
 28. A method of detecting a cytokine in asample comprising contacting the sample with a sorbent according toclaim 26 under conditions suitable for binding the cytokine onto thesorbent and detecting the cytokine bound to the sorbent.